Antisense oligonucleotide (ASO), including DNA, RNA and ribozymes, represent a potential new class of anti-viral/anti-cancer therapeutics. Due to the exquisite specificity of these compounds, they are unlikely to cause undesired side effects and therefore represent a substantial improvement over current ineffective and/or highly toxic anti-viral/anti- cancer drugs. The effectiveness of antisense (AS) mediated gene inhibition is influenced chiefly by factors that affect the ability of these agents to associate with their target. Key among these are, the availability of the target site for interaction with ASO, and the extent of cellular uptake of the antisense oligonucleotide. Target site availability is a function of both the structure of the RNA, as well as other ill defined cellular influences, including nucleic acid binding factors and RNA processing reactions. As a results, prediction of accessibility is beyond our present capability. Presently, accessibility of individual sites is evaluated empirically. This is not suitable for locating one (or a few) optimal site(s) that may be present on an individual mRNA. Specific aims 1 and 2 address the issue of target site accessibility by providing a method to locate optimal sites, on any RNA, for achieving maximal antisense mediated gene inhibition. This method uses a genetic scheme to examine all sites simultaneously, therefore no prior knowledge of the mRNA structure or the potential effects of other unknown cellular influences, is required. Specific Aim 1) Develop a rapid method for generating hammerhead ribozyme gene libraries. Specific Aim 2) Identify optimal sites for antisense mediated inhibition of tat, by in vivo expression of anti-tat ribozyme gene libraries. To overcome the other major obstacle to antisense efficiency, (i.e., poor uptake) we will examine two different polymer based carriers for their ability to increase cellular uptake of antisense ribozymes. Specific Aim 3) Examination of cationic and HPMA based polymers as carriers for the efficient delivery of antisense ribozymes. We will characterize the kinetics and extents of uptake by cultured mammalian cells of carrier and non-carrier bound ribozymes. Using fluorescence microscopy and fluorescence resonance energy transfer techniques we will determine if the intracellular fate of the ribozyme is altered by delivery in association with the polymer carriers. Finally, in vivo antisense activity will be examined and correlated with the results form the uptake and fluorescence experiments. These studies will allow determination of the effectiveness of polymer based carriers for increasing uptake and efficacy of antisense therapeutics.